title: Evaluation of UDP-GlcN derivatives for selective labeling of 5-(hydroxymethyl)cytosine.
abstract: 5-(hydroxymethyl)cytosine (5-hmC) is a newly identified oxidative product of 5-methylcytosine (5-mC) in the mammalian genome, and is believed to be an important epigenetic marker influencing a variety of biological processes. In addition to its relatively low abundance, the fluctuation of 5-hmC levels over time during cell development poses a formidable challenge for its accurate mapping and quantification. Here we describe a specific chemoenzymatic approach to 5-hmC detection in DNA samples by using new uridine 5'-diphosphoglucosamine (UDP-GlcN) probes. Our approach requires modification of the glucose moiety of UDP-Glc with small amino groups and transfer of these glucose derivatives to the hydroxy moiety of 5-hmC by using T4 phage glucosyltransferases. We evaluated the transfer efficiencies of three glucosyltransferases (wild-type α- and β-GTs and a Y261L mutant β-GT) with five different UDP-Glc derivatives containing functionalized groups for subsequent bioconjugation and detection. Our results indicate that UDP-6-N3 -Glc, UDP-6-GlcN, and UDP-2-GlcN can be transferred by β-GT with efficiencies similar to that seen with the native UDP-Glc cofactor. 6-N3 -Glc- and 6-GlcN-containing oligonucleotides were selectively labeled with reactive fluorescent probes. In addition, a 2 kb DNA fragment modified with 2-GlcN groups was specifically detected by use of a commercially available antiglucosamine antibody. Alternative substrates for β-GT and correlated glycosyltransferases might prove useful for the study of the function and dynamics of 5-hmC and other modified nucleotides, as well as for multiplex analysis.
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938 (USA).
authors.forename: Nan
authors.initials: N
authors.lastname: Dai
authors.forename: Jurate
authors.initials: J
authors.lastname: Bitinaite
authors.forename: Hang-Gyeong
authors.initials: HG
authors.lastname: Chin
authors.forename: Sriharsa
authors.initials: S
authors.lastname: Pradhan
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D000906:Antibodies / Q000276:immunology; D017122:Bacteriophage T4 / Q000201:enzymology; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005944:Glucosamine / Q000737:chemistry* / Q000276:immunology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D051379:Mice; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling; D013379:Substrate Specificity; D014500:Uracil Nucleotides / Q000737:chemistry*
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D014500:Uracil Nucleotides; C035436:uridine 5'-tetraphosphate; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D005964:Glucosyltransferases; D005944:Glucosamine
keywords: (hydroxymethyl)cytosine; DNA; chemical labeling; glucosamines; glycosylation